Lactobacillus mucilaginosus UP-1 and application thereof in improvement of bone metabolism and promotion of growth

Abstract

The invention relates to fermented lactobacillus mucus UP-1 and application thereof to improvement of bone metabolism and promotion of growth, and belongs to the technical field of microorganisms, the classification name of the fermented lactobacillus mucus UP-1 is Limosilicobacterium fermentum UP-1, the preservation unit of the fermented lactobacillus mucus UP-1 is China General Microbiological Culture Collection Center, and the preservation number of the fermented lactobacillus mucus UP-1 is CGMCC No.33076; the preservation time is December 16, 2024. The fermented lactobacillus mucus UP-1 provided by the invention can promote the increase of the body length and the thighbone length diameter by increasing the levels of insulin-like growth factor binding protein-3 (IGFBP3), growth hormone (GH) and insulin-like growth factor (IGF-1) in serum, improve the density of bone trabecula and the level of beneficial metabolite short-chain fatty acid, improve the bone metabolism level, promote the growth of a human body and promote the growth of a human body. The health development of bones is promoted, so that the purposes of promoting growth and development and improving nutrient absorption are achieved, and more scientific diet intervention and precise probiotic development are facilitated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to fermented Lactobacillus mucilaginosus UP-1 and application thereof in improving bone metabolism and promoting growth. Background Art

[0002] Growth and development are governed by different genetic, nutritional, and environmental factors that act systemically on the endocrine system and growth plate. A large number of studies have revealed the important role of the gut microbiota in the growth and development process. Whether it is physical growth, nervous system maturation, or immune system reinforcement, the gut microbiota should be regarded as an "organ" that has an important impact on growth and development. Its connection with growth and development provides a unique way to improve health and prevent disease. Compared with mice with normal flora, the growth and development of germ-free mice are reduced, and the longitudinal growth of bones is improved when the gut flora is recolonized to germ-free mice. At present, the molecular mechanism by which the gut microbiota promotes bone growth is still unclear. In addition to regulating the host growth hormone (GH) / insulin-like growth factor-1 (IGF-1) axis to mediate the host growth and development process, another possible explanation is that gut microbes help digest macronutrients. These studies highlight the positive role of gut microbes in the hormonal and nutritional regulation of bone growth and body growth.

[0003] As an important intestinal microbial "regulator", probiotics are widely used in the fields of health food, dairy processing, animal husbandry, and live bacterial drug development. Its role in promoting the growth of the body is mainly reflected in improving the intestinal environment, enhancing nutrient absorption, regulating the immune system, and inhibiting the reproduction of pathogens. Studies have found that the cross-regulation of the intestinal-bone axis is related to the fact that probiotics can directly or indirectly produce short-chain fatty acids (SCFAs) and other beneficial microbial metabolites. These metabolites act on the liver and adipose tissue to induce the production of IGF-1, and IGF-1 acts on bone cells to affect linear growth, bone mass and mineralization. At the same time, metabolites can directly act on bones and muscles to induce local IGF-1 production. Another study also confirmed this point. Supplementation with Lactobacillus rhamnosus LGG can indirectly promote the production of SCFAs by intestinal microorganisms. SCFAs further selectively support the development of peripheral regulatory T cells and stimulate bone formation, thereby improving growth and development performance. More importantly, SCFAs can also play a role in nutritional regulation by stimulating intestinal blood flow and electrolyte uptake. About 10% of the total energy intake of humans comes from SCFAs absorption. In addition, probiotics stimulate the activity of immune cells (such as macrophages, T cells, and B cells) by interacting with the gut-associated lymphoid tissue (GALT), promote the secretion of anti-inflammatory cytokines (such as IL-10), and inhibit the excessive production of proinflammatory cytokines (such as IL-6 and TNF-α). This immunomodulatory effect helps to reduce inflammatory responses, reduce energy consumption, and create good conditions for the normal growth of the body. Obviously, supplementing with probiotics is one of the effective strategies to promote host growth. However, there are few studies evaluating the effects of probiotics on normal growth and development, and the specific effects of different strains, dose-response relationships, and efficacy in specific populations are still unclear.

[0004] Therefore, developing a probiotic preparation that can promote normal children has very important scientific significance and economic value. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a fermented mucus lactobacillus preparation and application which can promote digestion and absorption, growth and development, and improve bone metabolism. The strain can effectively promote the physical growth of the body, increase the level of beneficial metabolites of the body, improve the level of bone metabolism, promote the healthy development of bones, and have a positive effect on the growth and development of the body and nutrient absorption.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: 1. Isolation, identification and safety assessment of probiotics The present invention isolates a strain of fermented mucus lactobacillus ( Limosilactobacillus fermentum). Phenotypic identification, molecular biological identification, and phylogenetic analysis were used to identify the strain as Lactobacillus mucosa fermentans, and its hemolytic activity, antibiotic sensitivity, and bacterial pathogenicity were evaluated to confirm its safety. In addition, its tolerance to gastric and intestinal fluids was tested to determine that it could reach the intestine alive.

[0007] 2. Growth-promoting effects of probiotics on different female and male mice The present invention uses female and male mice as animal models to explore the effects of probiotics on the body's growth performance and digestion and absorption, statistics the body length changes and body length growth rate of mice, detects changes in mouse serum growth indicators to evaluate growth status, and evaluates digestion and absorption levels in combination with changes in short-chain fatty acids in the mouse intestine.

[0008] The present invention provides a fermented Lactobacillus mucilaginosus UP-1 ( Limosilactobacillus fermentum ), the depository is the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit number is: CGMCC No. 33076; the deposit time is: December 16, 2024.

[0009] The present invention also provides a preparation comprising fermented Lactobacillus mucilaginosus UP-1.

[0010] The preparation containing fermented Lactobacillus mucilaginosus UP-1 is one or more of food raw materials, probiotic powder, food additives, feed additives, medicines, liquid drinks, foods or health products.

[0011] The preparation containing fermented Lactobacillus mucilaginosus UP-1 is a solid or liquid preparation; wherein, when the preparation is a liquid preparation, it contains 1.0 × 10 5 CFU / ml~1.0 × 10 12 CFU / ml amount of fermented Lactobacillus mucilaginosus UP-1; when the preparation is in solid form, it contains 1.0×10 5 CFU / g~1.0×10 12 CFU / g amount of fermented Lactobacillus mucilaginosus UP-1.

[0012] The preparation containing fermented mucus lactobacillus UP-1 also contains auxiliary materials, and the auxiliary materials include one or more of edible vegetable oil, medium-chain triglycerides, inulin, oligofructose, resistant dextrin, oligogalactose, skim milk powder, desalted whey powder, lactoferrin, casein phosphopeptide, vitamins, calcium agents or hydrolyzed egg yolk powder.

[0013] The present invention also provides a product for promoting digestion and absorption, growth and development, and improving bone metabolism, comprising the fermented Lactobacillus mucilaginosus UP-1 or the culture.

[0014] Preferably, the product is food, medicine or health product.

[0015] Preferably, the product contains no less than 1×10 9 CFU / mL or 1×10 9 CFU / g of fermented Lactobacillus mucilaginosus UP-1.

[0016] In the embodiment, promoting the growth of the subject comprises: one or more of promoting an increase in the subject's body length, and promoting an increase in the subject's body length growth rate.

[0017] In the embodiment, the subject is a mouse. Compared with the prior art, the present invention has the following beneficial effects: The fermented mucus lactobacillus UP-1 ( Limosilactobacillus fermentum ) can increase the levels of insulin-like growth factor binding protein-3 (IGFBP3), growth hormone (GH) and insulin-like growth factor (IGF-1) in serum, promote the increase of body length and femur long diameter, increase trabecular bone density and the level of beneficial metabolites short-chain fatty acids, improve bone metabolism, and promote healthy bone development, thereby achieving the purpose of promoting growth and development and improving nutrient absorption, and contributing to more scientific dietary intervention and the development of precision probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The colony morphology of Lactobacillus muciferus UP-1 is shown.

[0019] Figure 2 The microscopic morphology of Lactobacillus mucilaginosus UP-1 under scanning electron microscope is shown.

[0020] Figure 3 The phylogenetic tree of Lactobacillus muciferum UP-1 is shown.

[0021] Figure 4 The chromosome genome circle map of Lactobacillus mucilaginosus UP-1 is shown.

[0022] Figure 5 The gastrointestinal fluid tolerance of Lactobacillus mucilaginosus UP-1 was shown.

[0023] Figure 6 Hemolytic assay of Lactobacillus mucilaginosus UP-1 is shown.

[0024] Figure 7 Shows the effect of fermented Lactobacillus mucilaginosus UP-1 on the body length of mice. Figure A is female, Figure B is male, * indicates P <0.05, ** indicates P <0.01.

[0025] Figure 8The effect of fermented Lactobacillus mucilaginosus UP-1 on the body length growth rate of mice, Figure A is female, Figure B is male, ** indicates P <0.01.

[0026] Fig. 9 The effect of fermented Lactobacillus mucilaginosus UP-1 on the long diameter of the femur of mice, Figure A is female, Figure B is male, * indicates P <0.05, **** indicates P <0.0001.

[0027] Fig.10 The effect of fermented Lactobacillus mucilaginosus UP-1 on mouse growth factor binding protein-3 is shown. Figure A is female, Figure B is male, and *** indicates P <0.001.

[0028] Fig.11 The effect of fermented Lactobacillus mucilaginosus UP-1 on growth hormone in mice is shown. Figure A is for females and Figure B is for males. ** indicates P <0.01, **** indicates P <0.0001.

[0029] Fig.12 The effect of fermented Lactobacillus mucilaginosus UP-1 on growth factors in mice is shown. Figure A is for females and Figure B is for males. ** indicates P <0.01, *** indicates P <0.001.

[0030] Fig.13 The effect of fermented Lactobacillus mucilaginosus UP-1 on the amino-terminal propeptide of type I procollagen in mice is shown in Figure A for females and Figure B for males. * indicates P <0.05, *** indicates P <0.001.

[0031] Fig.14 The effect of fermented Lactobacillus mucilaginosus UP-1 on the ratio of amino-terminal propeptide of type I procollagen to cross-linked carboxyl-terminal peptide of type I collagen in mice is shown in Figure A for females and Figure B for males. **** indicates P <0.0001.

[0032] Fig.15 Shows the effect of fermented Lactobacillus mucilaginosus UP-1 on the osteoclast differentiation factor RanKL in mouse bone marrow. * indicates P <0.05, **** indicates P <0.0001.

[0033] Fig.16The effect of fermented Lactobacillus mucinus UP-1 on bone mass and bone metabolism in female mice is shown. A is the 3D reconstruction of the femur of mice in the fermented Lactobacillus mucinus UP-1 group, B is the 3D reconstruction of the femur of mice in the control group, C is the bone volume fraction, and * indicates P <0.05.

[0034] Fig.17 The effect of fermented Lactobacillus mucinus UP-1 on bone mass and bone metabolism in male mice is shown. A is the 3D reconstruction of the femur of mice in the fermented Lactobacillus mucinus UP-1 group, B is the 3D reconstruction of the femur of mice in the control group, C is the bone volume fraction, and * indicates P <0.05.

[0035] Fig.18 Shows the effect of fermented Lactobacillus mucilaginosus UP-1 on pro-inflammatory factors in mouse colon. * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001.

[0036] Fig.19 Shows the effect of fermented Lactobacillus mucilaginosus UP-1 on the level of short-chain fatty acids in the intestine of female mice. * indicates P <0.05, ** indicates P <0.01.

[0037] Fig. 20 Shows the effect of fermented Lactobacillus mucilaginosus UP-1 on the level of short-chain fatty acids in the intestine of male mice. * indicates P <0.05, ** indicates P <0.01.

[0038] Biological Deposit A fermented Lactobacillus mucilaginosus UP-1 ( Limosilactobacillus fermentum ) is named as: Lactobacillus fermentans Limosilactobacillus fermentum ; Depository: General Microbiology Center of China Microbiological Culture Collection Administration, deposit number: CGMCC No. 33076; Deposit date: December 16, 2024; Deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; The test result of this strain is survival. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description progresses. However, these examples are exemplary only and do not constitute any limitation on the scope of the present invention. The methods, ingredients and dosages involved in the following embodiments, unless otherwise specified, are conventional methods known to those skilled in the art. It is worth noting that the raw materials used in the present invention are all common commercially available products, among which MRS liquid culture medium is purchased from Beijing Luqiao Technology Co., Ltd.; fermented mucus lactobacillus UP-1 is provided by the Key Laboratory of Dairy Biotechnology and Engineering of the Ministry of Education, Inner Mongolia Agricultural University. In the embodiments of the present invention, the mammalian BALB / c mouse is a conventional model organism in the prior art, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0040] Example 1 Fermented Lactobacillus mucilaginosus UP-1 ( Limosilactobacillus fermentum ) isolation and identification: (1) Separation and purification Lactobacillus mucilaginosus UP-1 was isolated from breast milk of healthy women in 2018. 0.5 mL of the sample was added to 5 mL of MRS liquid culture medium and cultured aerobically at 37°C for 24 h to obtain the cultured sample. The sample was gradually diluted to an appropriate gradient (10 6 ~10 7 CFU / mL), 100 μL of gradient dilutions were spread on MRS solid medium, and aerobically cultured at 37°C for 48 h to obtain colonies; colonies with typical characteristics of fermentative mucus Lactobacillus on MRS solid medium were selected according to colony shape, size, edge and transparency, and colonies were picked with an inoculation loop for streaking on MRS solid medium, and aerobically cultured at 37°C for 24 h, and then the above steps were repeated until pure colonies were obtained.

[0041] (2) Passaging and cryopreservation Use an inoculation loop to scrape the colonies on the MRS solid culture medium and inoculate them into 10 mL of MRS broth medium. Culture at 37°C for 24 h, then mix the sterilized skim milk and the bacterial solution in a 1:1 volume ratio, mix well, and store the strain at -80°C.

[0042] (3) Identification of strains Fermented mucus Lactobacillus UP-1 was identified using the FMIC-QO01-001-2015 microbiological detection bacterial multiphase identification test method. On MRS medium, aerobic culture was carried out at 37 °C for 24 h, and the colony morphology was observed. Fermented mucus Lactobacillus UP-1 showed white, round, moist, opaque, and neatly edged colonies ( Figure 1), and the bacteria are rod-shaped, 0.4-0.6 μm× 0.7-1.6 μm, arranged singly or in pairs, Gram-positive ( Figure 2 ).

[0043] (4) Phylogenetic analysis of strains Fresh fermented mucus lactobacillus UP-1 bacterial solution was used as a template in the PCR amplification system for PCR amplification, the genome of strain UP-1 was extracted using a bacterial genome extraction kit, and the extracted genome of strain UP-1 was used as a template for amplification to obtain 16S rRNA of strain UP-1; the 16S rDNA of strain UP-1 was subjected to homologous sequence alignment using BLAST software in the NCBI database, and the alignment results were compared using MEGA software, and a phylogenetic tree was constructed using the adjacent joining method, and the similarity was repeatedly calculated 1000 times. The isolated and identified strain was named fermented mucus lactobacillus UP-1, and the sequence of the 16SrRNA gene was obtained by 16S rRNA gene sequencing as shown in SEQ ID No. 1, specifically: GTTGGCCCAA TTGATTGATG GTGCTTGCAC CTGATTGATT TTGGTCGCCA ACGAGTGGCG GACGGGTGAG TAACACGTAG GTAACCTGCC CAGAAGCGGG GGACAACATT TGGAAACAGA TGCTAATACC GCATAACAAC GTTGTTCGCA TGAACAACGC TTAAAAGATG GCTTCTCGCT ATCACTTCTG GATGGACCTG CGGTGCATTA GCTTGTTGGT GGGGTAACGG CCTACCAAGG CGATGATGCA TAGCCGAGTT GAGAGACTGA TCGGCCACAA TGGGACTGAG ACACGGCCCA TACTCCTACG GGAGGCAGCA GTAGGGAATC TTCCACAATG GGCGCAAGCC TGATGGAGCA ACACCGCGTG AGTGAAGAAG GGTTTCGGCT CGTAAAGCTC TGTTGTTAAA GAAGAACACG TATGAGAGTA ACTGTTCATA CGTTGACGGT ATTTAACCAG AAAGTCACGG CTAACTACGT GCCAGCAGCC GCGGTAATAC GTAGGTGGCA AGCGTTATCC GGATTTATTG GGCGTAAAGA GAGTGCAGGC GGTTTTCTAA GTCTGATGTG AAAGCCTTCG GCTTAACCGG AGAAGTGCAT CGGAAACTGG ATAACTTGAG TGCAGAAGAG GGTAGTGGAA CTCCATGTGT AGCGGTGGAA TGCGTAGATA TATGGAAGAA CACCAGTGGC GAAGGCGGCT ACCTGGTCTG CAACTGACGC TGAGACTCGA AAGCATGGGT AGCGAACAGG ATTAGATACC CTGGTAGTCC ATGCCGTAAA CGATGAGTGC TAGGTGTTGG AGGGTTTCCG CCCTTCAGTG CCGGAGCTAA CGCATTAAGC ACTCCGCCTG GGGAGTACGA CCGCAAGGTT GAAACTCAAA GGAATTGACG GGGGCCCGCA CAAGCGGTGG AGCATGTGGT TTAATTCGAA GCTACGCGAA GAACCTTACC AGGTCTTGAC ATCTTGCGCC AACCCTAGAG ATAGGGCGTT TCCTTCGGGA ACGCAATGAC AGGTGGTGCA TGGTCGTCGT CAGCTCGTGT CGTGAGATGT TGGGTTAAGT CCCGCAACGA GCGCAACCT TGTTACTAGT TGCCAGCATT AAGTTGGGCA CTCTAGTGAG ACTGCCGGTG ACAAACCGGA GGAAGGTGGG GACGACGTCA GATCATCATG CCCCTTATGA CCTGGGCTAC ACACGTGCTA CAATGGACGG TACAACGAGT CGCGAACTCG CGAGGGCAAG CAAATCTCTT AAAACCGTTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGTC GGAATCGCTA GTAATCGCGG ATCAGCATGC CGCGGTGAAT ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGA GAGTTTGTAA CACCCAAAGT CGGTGGGGTA ACC UP-1 phylogenetic tree Figure 3 As shown, it was identified as Lactobacillus mucimulentus ( Limosilactobacillus fermentum ) and is a species of bacteria permitted for use in food in the List of Bacteria Permitted for Use in Food. The results of whole genome sequencing and analysis of the strain showed that the UP-1 genome contained 1 chromosome (2,253,013 bp) and 1 plasmid (42,454 bp), with a total sequence length of 2,281,393 bp and a GC content of 50.82 ( Figure 4 ).

[0044] Example 2 Characteristics of Lactobacillus mucilaginosus UP-1 (1) Artificial gastric juice and artificial intestinal juice tolerance test Preparation of artificial gastric juice: 40ml sterilized PBS normal solution + 3.0g / L pepsin (0.3%), then adjust the pH to 2.5 (adjust with 0.1mol / L HCL), filter with a 0.22μm filter membrane into a sterilized empty conical bottle for later use; Preparation of artificial intestinal fluid: 40 ml of sterile PBS normal solution + 1.0 g / L trypsin (0.1%) + 1.8% bile salt, then adjust the pH to 8.0 (adjust with 0.1 mol / L NaOH), filter with a 0.22 microfiltration membrane into a sterilized empty conical flask and set aside.

[0045] This study tested the tolerance of UP-1 to digestion in artificial gastric juice at pH 2.5 for 3 hours and artificial intestinal juice at pH 8.0 for 8 hours. After culturing UP-1 strain for 24 hours, the bacteria were collected by centrifugation, washed twice with PBS, and vibrated with 5 mL of PBS as the original bacterial solution. The original bacterial solution was diluted and counted as the value of 0h; 500 microliters of the original bacterial solution was taken into a test tube containing 4.5 mL of gastric juice, placed in a 37℃ water bath for 3 hours, and counted; 500 microliters of the bacterial solution after 3 hours of culture was taken into a test tube containing 4.5 mL of intestinal juice, placed in a 37℃ water bath for 8 hours, and counted.

[0046] Results Figure 5 The survival rate of fermented Lactobacillus mucilaginosus UP-1 in artificial gastric juice of pH 2.5 for 3 hours was 73.26%, and then it was transferred to artificial intestinal juice of pH 8.0 for 8 hours, and its survival rate was 78.20%, indicating that fermented Lactobacillus mucilaginosus UP-1 has a strong survival ability in artificial gastrointestinal juice.

[0047] Example 3 Safety evaluation of fermented Lactobacillus mucilaginosus UP-1 (1) Drug sensitivity testing This experiment was conducted according to EFSA 5206-2018 "Guidelines for the microbial characteristics of feed additives or fermented products" 2.2.1 Antimicrobial susceptibility test methods to test the resistance of strains to ampicillin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol. The resistance of strains is expressed by the minimum inhibitory concentration (MIC).

[0048] The results of MIC test are shown in Table 1 . UP-1 was sensitive to ampicillin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol.

[0049] Table 1 MIC values ​​and drug susceptibility of fermented Lactobacillus mucilaginosus UP-1 ; (2) Antibiotic resistance genes and virulence genes The FMIC-QO01-024-2020 microbiological detection method for detecting bacterial antibiotic resistance genes and virulence genes was used to detect the antibiotic resistance genes and virulence genes of fermented mucus lactobacillus UP-1. In terms of antibiotic resistance genes, the database CARD and software RGI were used, and the Perfect and Strict algorithms were used as screening criteria, and no antibiotic resistance-related genes were detected. With sequence similarity ≥90% and sequence coverage ≥60% as screening criteria, no antibiotic resistance-related genes were detected. In terms of virulence genes, setA, whose sequences were experimentally verified in the database, was used as the analysis data set, and sequence similarity ≥80% and sequence coverage ≥70% were used as screening criteria, and no virulence-related genes were detected. With sequence similarity ≥90% and sequence coverage ≥60% as screening criteria, no virulence-related genes were detected. It shows that fermented mucus lactobacillus UP-1 does not contain exogenous antibiotic resistance genes and virulence genes, and is safe to eat.

[0050] (3) Hemolytic test Listeria innocua ( Listeria innocua ) CICC 10417, and Staphylococcus aureus was used as the positive control bacteria ( Staphylococcus aureus ) CICC 10417, which were purchased from Beijing Ketuo Hengtong Biotechnology Co., Ltd. According to the SH-QO01-008-2019 microbiological detection of microbial strain hemolysis detection method, UP-1 was tested for hemolysis. Figure 6 As shown, UP-1 is a white smooth colony with no hemolysis on the blood plate, which indicates that the target strain has no obvious pathogenicity and is relatively safe.

[0051] (4) Animal pathogenicity testing According to the Technical Guidelines for Safety Testing and Evaluation of Bacteria for Health Food Raw Materials (2020 Edition), Appendix A, Pathogenicity Test Methods for Bacteria for Health Food Raw Materials, the animal pathogenicity was tested. The results of the intraperitoneal injection test are shown in Table 2 (male) and Table 3 (female). There was no significant difference in the initial and final weights of male / female mice between the culture group and its corresponding control group ( p >0.05), that is, during the experimental period, the fermentation of Lactobacillus mucinus UP-1 culture had no effect on the body weight of male / female mice, and the fermentation of Lactobacillus mucinus UP-1 culture had no adverse effect on the general health of the animals, and no toxic reactions or deaths were observed in the test mice (Table 4).

[0052] Table 2 Effects of fermented Lactobacillus mucilaginosus UP-1 culture on body weight of male mice (intraperitoneal injection) ; Table 3 Effects of fermented Lactobacillus mucilaginosus UP-1 culture on body weight of female mice (intraperitoneal injection) ; Table 4 Acute toxicity of fermented Lactobacillus mucilaginosus UP-1 culture to mice (intraperitoneal injection) .

[0053] Example 4 A fermented Lactobacillus mucilaginosus UP-1 bacterial powder is prepared by a conventional bacterial powder preparation method, and the steps are as follows: (1) Activation of bacterial strains The strains stored at -80°C were inoculated into the corresponding liquid culture medium sterilized at 121°C for 15 min, and cultured anaerobically at 37°C for 18-24 h. The activated strains were obtained by subculturing 1-2 times.

[0054] (2) Preparation of seed solution Each activated bacterial strain in step (1) is inoculated into MRS liquid culture medium and cultured anaerobically at 37°C until the pH value reaches 4.5-4.8; (3) Inoculation and fermentation The seed liquid of step (2) was inoculated into MRS liquid culture medium at 1‰, and fermented for 18 hours under controlled fermentation conditions; the fermentation conditions were controlled as follows: constant temperature culture at 30°C in the early stage of fermentation, and natural fermentation until the pH value was 5.0; then the fermentation temperature was adjusted to constant temperature culture at 37°C, and the pH value was controlled to be maintained at 6.0, and aerobic fermentation was maintained.

[0055] In the above process, pH is controlled by adding a neutralizing agent, and the neutralizing agent is NaOH.

[0056] (4) Freeze drying a. Bacterial cell concentration: The high-density fermentation broth was centrifuged at 12,000 g to concentrate the cells; b. Add protective agent: Add 5 times the protective agent solution to the bacterial concentrate; the protective agent solution is composed of 14 Kg of skim milk, 10 Kg of lactose, 1.1 Kg of vitamin C, 0.8 Kg of sodium glutamate and 1000 L of distilled water.

[0057] c. Drying: The bacterial suspension after adding the protective agent is freeze-dried to obtain freeze-dried bacterial powder, and the total number of viable bacteria in the bacterial powder reaches 1.0×10 11 CFU / g and above.

[0058] The total viable bacteria content of the fermented Lactobacillus mucilaginosus UP-1 powder is 1×10 11 CFU / g.

[0059] The UP-1 bacterial powder prepared in this embodiment can be used for food, medicine, health care products or animal feed. The food can be ordinary food or health care food such as fermented milk, cheese, milk-containing beverages, solid beverages, milk powder, etc. Preferably, in the food, the recommended dosage of fermented Lactobacillus mucilaginosus UP-1 for human use can be 1.0×10 3 CFU~1.0 ×10 10 CFU / kg body weight / day, more preferably 1.0×10 4 CFU~1.0×10 9 CFU / kg body weight / day.

[0060] Example 5 Functional characteristics of a fermented Lactobacillus mucilaginosus UP-1 powder in promoting growth: 1. Experimental methods (1) Grouping Forty-eight 3-week-old BALB / c mice were selected. After all mice were adaptively fed with basic feed for one week, they were randomly divided into a control group and a fermented Lactobacillus mucosa UP-1 bacterial powder group, with 24 mice in each group, half of which were male and half were female. The UP-1 bacterial powder treatment group was gavaged with 1×10 viable bacteria per day. 9 The experimental animals were housed in an SPF barrier environment with a temperature of 20-26°C and a humidity of 30%-70%, and were allowed to eat and drink freely.

[0061] (2) Phenotypic indicators The mental state and activity of the mice were observed at a fixed time every day, the water intake and food intake of each group of mice were monitored and recorded, and the body weight and length were calculated once a week.

[0062] (3) Blood collection Blood samples were collected from mice in the 6th week of the experiment: the mice were anesthetized and blood was collected using the retrobulbar sinus bleeding method (RBB). Blood was collected using a disposable blood collection tube containing separation gel / coagulant and centrifuged at 4°C and 3000 × g for 15 min. The supernatant was collected and dispensed into 200 µL sterile EP tubes and stored at -80°C.

[0063] (4) Detection of growth indicators The effects of UP-1 bacterial powder on host growth and development were evaluated using a kit (ELISA) to measure serum insulin-like growth factor (IGF-1), insulin-like growth factor binding protein-3 (IGFBP3), ghrelin, growth hormone (GH), type I procollagen amino-terminal propeptide (PINP), and type I collagen cross-linked carboxyl-terminal peptide (CTXI).

[0064] (6) Fecal collection and detection of short-chain fatty acids Fecal samples of mice were collected in the sixth week of the experiment: each mouse was placed in an autoclaved cage, and after the mouse defecated naturally, fresh feces ≥3 pieces were collected with sterile tweezers and placed in a 2 mL sterile EP tube, which was quickly frozen with liquid nitrogen and stored at -80 °C. After pretreatment of the fecal samples, liquid chromatography-mass spectrometry was used to quantitatively detect the extracted short-chain fatty acids.

[0065] 2. Experimental Results (1) Effect of fermented Lactobacillus mucilaginosus UP-1 on body length of mice After 6 weeks of intervention with UP-1 powder, the body length of mice was measured. Figure 7 As shown, compared with the control group (NC), UP-1 bacterial powder significantly increased the female mice ( Figure 7 A) and male mice ( Figure 7 B) (P<0.05), and the body length growth rate also increased significantly ( Figure 8 , P<0.05). The long diameter of the femur of mice was tested. The results are as follows Fig. 9 As shown, female mice ( Fig. 9 A) and male mice ( Fig. 9 The femur length of mice in the control group (B) was significantly higher than that of the control group, indicating that fermented Lactobacillus mucosa UP-1 powder can promote the growth of femur and body length in mice.

[0066] (2) Effects of fermented Lactobacillus mucilaginosus UP-1 on serum growth indicators in mice The level of insulin-like growth factor binding protein-3 (IGFBP3) is closely related to growth hormone (GH) and insulin-like growth factor (IGF-1). The three together form the GH-IGF-IGFBP axis, which is the core regulatory system for promoting the growth of children and adolescents. The level of IGFBP-3 is positively correlated with growth hormone and IGF-1, and is an important biomarker for diagnosing growth and development disorders. Therefore, this study detected the levels of IGF-1, IGFBP3, and GH in mouse serum. The results are as follows Figure 10-12 As shown, in female mice, the levels of IGF-1, IGFBP3, and GH in the serum of female UP-1 powder group mice were significantly higher than those of the female control group ( Fig.10 A, 11A, 12A, P <0.05), in male mice, the levels of IGF-1, IGFBP3, and GH in the serum of the male UP-1 powder group were also significantly higher than those in the male control group ( Fig.10 B, 11B, 12B, P <0.05). It is speculated that fermented Lactobacillus mucilaginosus UP-1 powder can promote the growth and development of mice by increasing the levels of IGF-1, IGFBP3, and GH in mouse serum through mediating the GH-IGF-IGFBP axis.

[0067] (3) Effects of fermented Lactobacillus mucilaginosus UP-1 on serum bone metabolites in mice Type I procollagen amino-terminal propeptide (PINP) and type I collagen cross-linked carboxyl-terminal peptide (CTXI) are two important biomarkers reflecting bone metabolism, representing the bone formation and bone resorption processes, respectively. PINP is a byproduct of type I collagen synthesis and is released into the blood during the synthesis of type I collagen by osteoblasts. It is a marker reflecting bone formation and is often used to evaluate bone regeneration and osteogenic activity. CTXI is a metabolite released into the blood when type I collagen is degraded, mainly derived from the bone resorption process. It is a marker reflecting bone resorption and is often used to evaluate osteoclast activity and bone loss. PINP levels can be used as a positive indicator of bone health during growth and development, and higher PINP means an active bone formation process. The PINP / CTXI ratio can be used to evaluate the balance of bone metabolism. A higher ratio indicates that bone formation is dominant, which is beneficial to growth and development. In the present study, the PINP level and PINP / CTXI ratio in the serum of female UP-1 bacterial powder group mice were significantly higher than those of the female control group ( Fig.13 A. 14A, P <0.05), the serum PINP level and PINP / CTXI ratio in the male UP-1 powder group were also significantly higher than those in the male control group ( Fig.13 B, 14B, P <0.05). This indicates that fermented Lactobacillus mucilaginosus UP-1 powder helps bone formation and promotes healthy bone development.

[0068] (4) Effect of fermented Lactobacillus mucilaginosus UP-1 on mouse osteoclast differentiation factor RanKL RANKL is a key factor in osteoclast formation. It binds to RANK (Receptor Activator of Nuclear Factor κ B) on osteoclast precursor cells and induces their differentiation into mature osteoclasts. Excessive expression of RANKL leads to increased bone resorption, thus causing osteoporosis. Fig.15 ), the level of osteoclast differentiation factor RanKL in the bone marrow of female UP-1 bacterial powder group mice was significantly lower than that of the female control group ( P <0.0001), and the level of osteoclast differentiation factor RanKL in the bone marrow of male UP-1 bacterial powder group mice was also significantly lower than that of the male control group ( P <0.05). This indicates that fermented Lactobacillus mucilaginosus UP-1 powder helps reduce bone resorption and the risk of osteoporosis, thereby promoting bone health.

[0069] (5) Effects of fermented Lactobacillus mucilaginosus UP-1 on femoral bone mass and bone metabolism in mice In order to evaluate the effect of fermented Lactobacillus mucilaginosus UP-1 on bone mass and bone metabolism in mice, this study used micro-CT to detect relevant parameters of mouse femur. Bone volume fraction (BV / TV) is a commonly used indicator for evaluating cortical bone and cancellous bone mass. For cancellous bone in the medullary cavity, this ratio can reflect the amount of trabecular bone in different samples. An increase in this value indicates that bone anabolism is greater than catabolism, and bone mass increases, and vice versa, which can indirectly reflect the state of bone metabolism. The results of this experiment are as follows Fig.16 (female), Fig.17 (Male) In the 3D reconstructed model, Lactobacillus mucilaginosus UP-1 female mice ( Fig.16 A) and male mice ( Fig.17 A) compared with the control group ( Fig.16 B and Fig.17 B) The femur is filled with a large number of trabeculae with narrow gaps. At the same time, UP-1 also significantly increased the bone volume fraction of female and male mice ( Fig.16 C&17C, P <0.05), indicating that fermentation of Lactobacillus mucilaginosus UP-1 can increase the body's trabecular bone density, strengthen bone quality, and promote bone metabolism.

[0070] (6) Effects of fermented Lactobacillus mucilaginosus UP-1 on pro-inflammatory factors in mouse colon Proinflammatory factors are core regulators of inflammatory responses and play an active role in maintaining the body's immune defense. However, their overexpression may lead to chronic inflammation and related diseases. Therefore, the proinflammatory factors tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6) in mouse colon were detected. The results are as follows Fig.18 As shown, the levels of TNF-α, IL-1β, and IL-6 in the colon of female UP-1 bacterial powder group mice were significantly lower than those in the female control group ( P <0.05), the levels of TNF-α, IL-1β, and IL-6 in the colon of the male UP-1 bacterial powder group were also significantly lower than those in the male control group ( P <0.05). This indicates that fermented Lactobacillus mucilaginosus UP-1 powder helps reduce the expression of pro-inflammatory factors, inhibit intestinal inflammation, and maintain intestinal health.

[0071] (7) Effects of fermented Lactobacillus mucilaginosus UP-1 on short-chain fatty acids in the intestine of mice Short-chain fatty acids (SCFAs) are the main products of anaerobic fermentation in the colon, as well as the main products of protein degradation and amino acid fermentation. They can reflect the digestion of feed and participate in the optimization of various physiological functions of the body and the prevention and treatment of various diseases. Therefore, this experiment detected and analyzed the levels of short-chain fatty acids in the intestines of each group of mice. The results of female mice are as follows: Fig.19As shown, the levels of acetic acid, propionic acid, and butyric acid in the intestine of female UP-1 bacterial powder group mice were significantly higher than those in the female control group ( P <0.05), the results for male mice are as follows Fig. 20 As shown, the levels of acetate and butyrate in the intestine of male UP-1 bacterial powder group mice were significantly higher than those of the male control group ( P <0.05). This result shows the promoting effect of fermented Lactobacillus mucilaginosus UP-1 on intestinal short-chain fatty acids. On the one hand, it reflects that UP-1 promotes the digestion of complex carbohydrates in the intestine, and on the other hand, it suggests the beneficial potential of UP-1 on the physiological function of the body.

[0072] In summary, the intake of fermented mucus lactobacillus UP-1 can improve the levels of insulin-like growth factor binding protein-3, growth hormone, insulin-like growth factor and growth hormone releasing peptide in serum, while improving the PINP / CTXI ratio and bone volume fraction, increasing trabecular bone density and the level of intestinal short-chain fatty acids, reducing the expression of osteoclast differentiation factor RanKL and pro-inflammatory factors, thereby improving bone metabolism, promoting healthy bone development, and having a positive effect on body growth and development, intestinal immunity and digestion and absorption. Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the scope of protection of the present invention. Simple modifications or equivalent substitutions made by ordinary technicians in the field to the technical solution of the present invention do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Fermented Lactobacillus mucilaginosus UP-1 (L imosilactobacillus fermentum ), characterized in that, The depository unit of the fermented mucus Lactobacillus UP-1 is the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 33076 and a deposit date of December 16, 2024.

2. A preparation, characterized in that The preparation comprises the fermented Lactobacillus mucilaginosus UP-1 according to claim 1.

3. The preparation according to claim 2, characterized in that The preparation is one or more of food raw materials, probiotic powder, food additives, feed additives, medicines, liquid drinks, foods or health products.

4. The preparation according to claim 2 or 3, characterized in that The preparation is a solid or liquid preparation; wherein the preparation comprises 1.0×10 5 CFU / mL ~ 1.0×10 12 CFU / mL or 1.0×10 5 CFU / g ~ 1.0×10 12 CFU / g amount of fermented Lactobacillus mucilaginosus UP-1.

5. The preparation according to claim 2 or 3, characterized in that The preparation further comprises auxiliary materials, which include one or more of edible vegetable oil, medium chain triglyceride, inulin, oligofructose, resistant dextrin, galacto-oligosaccharide, skim milk powder, desalted whey powder, lactoferrin, casein phosphopeptide, vitamins, calcium agent or hydrolyzed egg yolk powder.

6. A product, characterized in that The product comprises the fermented Lactobacillus mucilaginosus UP-1 according to claim 1.

7. The product according to claim 6, characterized in that The product is a food, medicine or health product, and the number of viable bacteria in the product is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g of fermented Lactobacillus mucilaginosus UP-1.

8. Use of the fermented Lactobacillus mucilaginosus UP-1 according to claim 1 in the preparation of products for improving bone metabolism and promoting body growth.

9. The use according to claim 8, wherein promoting body growth comprises one or more of promoting increase in body length and promoting increase in body length growth rate.

Images